2.9. Flow Cytometry
After treatments, the cells were collected and stained with Annexin V/propidium iodide (PI) (BD Biosciences, Franklin Lakes, NJ, USA), washed twice with cold PBS, and then resuspended in 1× binding buffer at a concentration of 1 × 106 cells/mL. Next, 100 µL of the cell suspension was transferred to a 5 mL culture tube, and 5 µL of PE Annexin V and 5 µL of PI were added. The cells were gently vortexed and incubated for 15 min at 25°C in the dark, and 400 µL of 1× binding buffer was added to each tube. Flow cytometry analysis was performed using the FACSCanto II system equipped with BD FACSDiva software (Becton-Dickinson, San Jose, CA, USA). Based on the features of cell necroptosis, PI-positive cells were considered as the target cells.